The present invention is directed towards the diagnosis of malignant cancer by detection of the mts-1 mRNA or the mts-1 protein encoded by the mts-1 gene. This invention further relates to cancer therapeutics. More particularly, this invention relates to compositions and methods for treating tumors, for example, by intercepting the interaction between Mts-1 and p53. The present invention is also directed to mammalian cell lines and tumors with high and low metastatic potential which have been developed to serve as useful model systems for in vitro and in vivo anti-metastasis drug screening.
Malignant cancer tumors shed cells which migrate to new tissues and create secondary tumors; a benign tumor does not generate secondary tumors. The process of generating secondary tumors is called metastasis and is a complex process in which tumor cells colonize sites distant from the primary tumor. Tumor metastasis remains the major cause of morbidity and death for patients with cancer. One of the greatest challenges in cancer research is to understand the basis of metastasis, i.e., what controls the spread of tumor cells through the blood and lymphatic systems and what allows tumor cells to populate and flourish in new locations.
The metastatic process appears to be sequential and selective, and is controlled by a series of steps since metastatic tumor cells: (a) are mobile and can disseminate from the original tumor; (b) are capable of invading the cellular matrix and penetrating through blood vessels; (c) possess ummunological markers, which allow them to survive passage through the blood stream, where they must avoid the immunologically active cytotoxic xe2x80x9cTxe2x80x9d lymphocytes; and (d) have the ability to find a favorable location to transplant themselves and successfully survive and grow.
Understanding the underlying molecular mechanisms in metastasis is the key to understanding cancer biology and its therapy. In clinical lesions, malignant tumors contain a heterogeneous population of cells, exhibiting a variety of biological characteristics, e.g., differential growth rates, cell surface structures, invasive capacities and sensitivity to various cytotoxic drugs. Researchers can take advantage of tumor heterogeneity factors, by identifying specific cell produced markers, which are unique for metastasis, to develop therapeutic regiments which do not rely only on surgical resection.
At this time it is not known whether the metastatic phenotype is under the regulation of a single or multiple gene(s), and whether these genes are independent or interrelated. However, a number of genes have become correlated with the formation and metastasis of tumors. For example, several normal cellular genes become oncogenes by incorporation into a retroviral genome. Due to the juxtaposition of new promoter elements, such incorporation frequently allows a potential oncogene to be expressed in inappropriate tissues or at higher levels than it normally would be expressed. It appears from work with tumorigenic retroviruses as well as other systems that misexpression of many cellular proteins, particularly those involved in the regulation of the cell cycle, cell mobility, or cell-cell interaction may lead to a cancerous phenotype.
The present invention discloses the human mts-1 gene and diagnosis of metastatic cancer by use of either antibodies directed against the mts-1 protein or mts-1 nucleic acid probes directed against mts-1 mRNA.
The mouse and rat mts-1 genes have been previously isolated under different names (i.e., 18A2, Linzer, et al., Proc. Natl. Acad. Sci. USA. 80:4271-4275, 1983; and p9Ka, Barraclough et al., J. Mol. Biol. 198:13-20, 1987) but no function or correlation of the mts-1 gene in metastatic cancer has been established prior to the present invention. Previous work has indicated that the protein now identified as the mts-1 protein is a calcium binding protein with homology to other calcium binding proteins such as, for example, the S-100 calcium protein, which are thought to have a role in cell growth (Linzer et al. supra; Jackson-Grusby et al., Nuc. Acids Res. 15:6677-6690, 1987; Goto et al., J. Biochem. 103:48-53, 1988). Other researchers suggest a role for p9Ka, later found to be identical to mts-1, in myoepithelial cell differentiation (Barraclough, et al., supra).
As determined uniquely by the present invention, the mammalian mts-1 gene is expressed at 10-100 fold higher levels in metastatic cells compared to non-metastatic cells and normal cells. Only a few types of normal cells, including lymphocytes and trophoblasts, express mts-1. Hence, the present invention demonstrates a surprising new property of mts-1: the misexpression of mts-1 within a cell or tissue is diagnostic of malignant cancer.
p53 is a tumor suppressor protein found in humans and other mammals (See, e.g., Harris, Science 262: 1980-1981, 1993). The wild-type p53 protein functions to regulate cell proliferation and cell death (also known as apoptosis). While the mechanism through which the wild-type p53 protein suppresses tumor cell growth is not completely defined, it is known that one key feature of the growth suppression is the capacity of p53 to act as a transcription factor (Farmer et al., Nature 358, 83-86, 1992; and Kern et al., Science 256, 827-830, 1992).
The nucleotide and amino acid sequences of human p53 have been reported by Zakut-Houri et al, EMBO J. 4: 1251-1255, 1985). The ability of p53 to bind DNA in a sequence-specific manner maps to amino acid residues 90-290 of human p53 (Pavletich et al, Genes Dev. 7: 2556-2564, 1993; and Wang et al, Genes Dev. 7: 2575-2586 1993); the tetramerization domain maps to amino acid residues 322-355 of human p53. The DNA binding-regulation domain maps to amino acid residues 364-393 of human p53 or to the corresponding region encompassing residues 361-390 of mouse p53 (Hupp et al., Cell 71: 875-886, 1992; and Halazonetis et al., EMBO J. 12: 1021-1028, 1993).
Inactivation of p53 is associated with more than half of all human tumors. The inactivation can occur by mutation of the p53 gene or through binding of p53 to viral or cellular oncogene proteins, such as the SV40 large T antigen and MDM2. Mutations of the p53 protein in most human tumors involve the sequence-specific DNA binding domain (Bargonetti et al., Genes Dev. 6: 1886-1898, 1992).
The present invention has further identified the tumor suppressor protein p53 as a target for the metastasis associated Mts1 protein.
The present invention is directed towards the diagnosis of metastatic cancer using an mts-1 nucleic acid or antibodies directed against the mts-1 protein. The present invention is also directed to isolated and purified mts-1 nucleic acids available for diagnostic tests and antibodies directed against the mammalian mts-1 proteins.
One aspect of the present invention is directed to a method for diagnosing metastatic cancer by contacting serum from an individual to be tested for such cancer with an antibody reactive with a mammalian mts-1 protein or an antigenic fragment thereof, for a time and under conditions sufficient to form an antigen-antibody complex, and detecting the antigen-antibody complex.
Another aspect of the present invention provides an isolated, recombinant nucleic acid encoding a human mts-1 gene or a fragment thereof, and replicable DNA sequences encoding an mts-1 polypeptide which express high levels of the mts-1 polypeptide. Isolated antisense mts-1 nucleic acids and expression vectors therefor are also contemplated by the present invention. Human mts-1 nucleic acids are preferred.
A further aspect of this invention is directed to isolated transformed host cells, such as prokaryotic microorganisms, yeast, insect cells and eukaryotic cells, containing mts-1 nucleic acids and replicable vectors containing DNA sequences encoding the mts-1 polypeptide.
A still further aspect of this invention provides isolated homogeneous mammalian mts-1 polypeptides and pharmaceutical compositions including such a mts-1 polypeptide or protein. Human mts-1 polypeptides are preferred.
Another aspect of this invention provides antibodies directed against an mts-1 polypeptide or any peptide, fragment or derivative of the mts-1 protein.
A further aspect of this invention is directed towards treatment of cancer by administering reagents, such as for example, anti-mts-1 antibodies capable of binding the mts-1 protein and antisense mts-1 nucleic acids capable of binding mts-1 sense mRNA.
One aspect of the invention provides compounds which interfere with the interaction between Mts-1 and p53 by binding to Mts-1 (i.e., binding-intercepting compounds).
Another embodiment of the present invention provides a method for intercepting the binding between p53 and Mts-1 in a subject by administering to the subject, an effective amount of a peptide which prevents the interaction between p53 and Mts-1 by binding to Mts-1. For example, one such peptide comprises the C-terminal region of p53 (amino acid 289-393 of human p53 or amino acid 289-390 of murine p53), in particular, amino acid 360-393 of human p53 or amino acid 360-390 of murine p53. Functional fragments or analogs of such peptides are also within the scope of the present invention. Another example of a binding-intercepting peptide comprises amino acid 1909-1937 of non-muscle myosin heavy chain or functional fragments of analogs thereof.
In one embodiment, the present invention provides methods of treating a tumor in a subject by administering to the subject, a therapeutically effective amount of a nucleic acid molecule coding for a peptide which prevents the binding of Mts-1 to p53.
In one embodiment, the present invention provides methods of treating a tumor in a subject by administering to the subject, a therapeutically effective amount of a peptide which prevents the binding of Mts-1 to p53.
In another embodiment, the present invention provides a method of treating a tumor in a subject by administering to the subject, a therapeutically effective amount of an antibody directed against Mts-1.
In still another embodiment, the present invention provides methods of treating a tumor in a subject by administering a therapeutically effective amount of an antisense DNA of an Mts-1 gene.
Yet another aspect of the present invention provides an animal model system of the metastatic process, including several eukaryotic cell lines and tumors expressing different levels of mts-1, which are derived from mouse and rat carcinomas. These cell lines and tumors may be re-introduced into mice or rats to produce primary tumors which metastasize to the lung, liver and kidneys with a characteristic frequency. Therefore, the present invention also provides a well controlled animal model system for testing pharmaceutical compositions suspected to have therapeutic utility for the treatment of metastatic cancer.